Cell growth substrates with tethered cell growth effector molecules

ABSTRACT

Disclosed are compositions with tethered growth effector molecules, and methods of using these compositions for growing cells and tissues. Growth effector molecules, including growth factors and extracellular matrix molecules, are flexibly tethered to a solid substrate. The compositions can be used either in vitro or in vivo to grow cells and tissues. By tethering the growth factors, they will not diffuse away from the desired location. By making the attachment flexible, the growth effector molecules can more naturally bind to cell surface receptors. A significant feature of these compositions and methods is that they enhance the biological response to the growth factors. The new method also offers other advantages over the traditional methods, in which growth factors are delivered in soluble form: (1) the growth factor is localized to a desired target cell population; (2) significantly less growth factor is needed to exert a biologic response. This method can be used as a means of enhancing the therapeutic use of growth factors in vivo and of creating surfaces which will enhance in vitro growth of difficult-to-grow cells such as liver cells.

BACKGROUND OF THE INVENTION

[0001] This invention concerns cell and tissue growth substrates, growthstimulation compositions, and methods for delivering growth factors tocells and tissues.

[0002] Long-term mammalian cell culture has been difficult to achieve.Many types of specialized cells plated on standard tissue cultureplastic dishes dedifferentiate, lose function, and fail to proliferate.There are many applications of mammalian cell culture that could benefitfrom methods or materials which enhance the long term stability ofdifferentiated mammalian cells in culture. These cells are currentlyused as sources of natural and engineered proteins and glycoproteins, inscreens for the effects of compounds on cell proliferation and function,and for implantation to supplement or replace cell function. Certaincells are particularly difficult to maintain in long term culture, suchas hepatocytes.

[0003] It would be especially useful if hepatocytes could be maintainedin long term culture. For example, in vitro toxicity testing ofingestible or orally administered compounds has been hampered by thefact that the liver converts many compounds into other chemical forms.These other forms may be toxic or have other effects. Thus completetesting of materials in cell culture must include the effects ofbiotransformations carried out by the liver. Using current methodology,it is difficult to grow normal liver cells in vitro beyond two to threecell divisions. The result is that in vitro testing does not reduce thenumber of animals needed because essentially all of the cells to be usedin vitro must come from direct isolation. A method of expanding livercells in vitro would make it feasible to use in vitro liver cellcultures to carry out biotransformations by applying the compound ofinterest directly to liver cells in culture. The supernate from theliver cell cultures could then be applied to other types of cells, suchas skin, lung, nerve, and bladder, to assess the effect of themetabolized compound of interest.

[0004] Studies have been conducted for a number of years to improve theviability, proliferation and differentiated function of eukaryotic cellscultured in vitro. One discovery has been the importance ofextracellular matrix and extracellular matrix molecules in maintainingcell function and allowing cell growth. These effects and methods ofusing matrix components for cell growth, have been described by, forexample. Jauregui et al., In Vitro Cellular & Developmental Biology 22:13-22 (1986), Kleinman et al., Analytical Biochemistry 166: 1-13 (1987),and Mooney et al., Journal of Cellular Physiology 151: 497-505 (1992).

[0005] Growth factors, such as epidermal growth factor (EGF),platelet-derived growth factor (PDGF), and transforming growth factors(TGFα, TGFβ), exert a broad mitogenic response. Growth factors and theireffects have been described in “Peptide Growth Factors and TheirReceptors I” M. B. Sporn and A. B. Roberts, eds. (Springer-Verlag, NewYork, 1990). In recognition of their importance, most cell and tissuegrowth compositions include growth factors, either as an additive or asa component of complex growth media. The use of growth factors in thismanner has certain drawbacks. For example, cells have a complex,nonlinear response to the concentration of growth factor in theirenvironment. Extended exposure to high growth factor concentrations maycause cells to lose responsiveness to the factor. For example, EGF, apotent mitogen for a wide variety of cell types and arguably thebest-characterized of the growth factors, when delivered in solubleform, is typically internalized by the cell, and the cell often respondsby a down-regulating the number of EGF receptors. This down-regulationcauses cells to lose responsiveness to EGF.

[0006] Growth factors have also been used in disappointingly fewclinical products, considering the range of effects they produce invitro. Translation of the mitogenic effects observed for the target cellin vitro to tissue growth in vivo is hampered by several issues. Forexample, the growth factors, when placed in a complex cellularenvironment, often end up stimulating the growth of competing cellswhich then overgrow the target cells. Researchers have attempted tosolve this problem by targeting delivery of factors at a specific site,but this approach is not always successful because soluble growthfactors can readily diffuse into the blood stream and away from thetarget site, exerting their effects elsewhere. This diffusion of growthfactors is also a problem because it increases the amount of growthfactor that must be used in order to have the desired local effect.Internalization of growth factors and loss of responsiveness to growthfactors is a particular problem for in vivo applications considering theamount of time cell growth must be stimulated to allow wound healing.

[0007] Another attempt to improve the longevity of growth factor effectsin vivo has been to incorporate growth factors in a slow releasematerial. Such a scheme still requires large amounts of growth factorand does not address the problem of competing cell growth due todiffusion of the growth factors. The large amount of growth factorsneeded for these cell and tissue growth methods is a particular problembecause growth factors are difficult and expensive to prepare.

[0008] It is therefore an object of the invention to provide a cell andtissue growth substrate that stimulates long-term target cell growth.

[0009] It is another object of the invention to provide a tissue growthscaffold for growth of a target tissue in vivo.

[0010] It is a further object of the invention to provide a method oflong-term cell and tissue growth in vitro, and to provide a method ofgrowing target tissue in vivo.

[0011] It is another object of the invention to provide an in vitrotissue analog for drug and toxicity testing, and a method of drug andtoxicity, testing using the tissue analog.

SUMMARY OF THE INVENTION

[0012] The methods and compositions described herein concern new celland tissue growth substrates. Growth effector molecules, includinggrowth factors and extracellular matrix molecules, are flexibly tetheredto a support medium and the combination is used to stimulate and supportcell and tissue growth. The most significant feature of thesecompositions is that they enhance the biological response to the growthfactor. The new compositions also offers other advantages over thetraditional growth methods, in which growth factors are delivered insoluble form: (1) the growth factor is localized to a desired targetcell population, and (2) significantly less growth factor is needed toexert a biologic response. In a preferred embodiment, multiple growthfactors and/or matrix materials are attached to a single core molecule,such as a star polymer. These compositions can be used as a means ofenhancing the therapeutic use of growth factors in vivo and of creatingsurfaces which will enhance in vitro growth of difficult-to-grow cellssuch as liver cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a graph of DNA synthesis in cells grown on anon-tethered substrate, with EGF present or absent from the growthmedium, plotting labeling index versus the presence or absence of EGF.The labeling index is the percentage of cells in a field that havestained nuclei.

[0014]FIG. 2 is a graph of DNA synthesis in cells grown with tethered(coupled) or adsorbed EGF, plotting labeling index versus tethered oradsorbed EGF. The labeling index is the percentage of cells in a fieldthat have stained nuclei.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Many problems with effective utilization of growth factors may beovercome if, instead of being delivered in soluble form, the growthfactors are immobilized on a solid substrate. This approach isattractive because some forms of insoluble matrix, such as crosslinkedcollagen sponges and bioresorbable polyester fabric, are used for manytypes of tissue regeneration to provide a template for tissue growth.The solid support need not be permanent, and thus the approach may beused for almost any tissue. Immobilization prevents the factor fromdiffusing away from the site and consequently allows a much more highlytargeted form of delivery than other methods. Besides this concentrationeffect, tethering has other powerful advantages, stemming from the waygrowth factors work. For example, when delivered in soluble form, EGF istypically internalized by the cell, and the cell often responds bydown-regulating the number of EGF receptors. However, evidence now showsthat the growth factor does not have to be internalized in order tostimulate cell growth. For example, Reddy et al., Biotechnology Progress10: 377-384 (1994), describes fibroblasts that remain responsive to EGFdespite their expression of internalization-deficient EGF receptors. Asdemonstrated by the following example, by allowing the target cell tobind EGF, but preventing the cell from internalizing the bound EGF, itis possible to circumvent the normal down-regulation of receptors thatoccurs in the presence of high concentrations of EGF. This offers twoadvantages: (1) it is possible to speed the rate of target cell growthin vivo because cells in contact with the surface bearing the growthfactor do not lose their responsiveness to EGF, and (2) considerablyless growth factor is required, because cells do not internalize anddegrade the growth factor. The method of attachment of the growth factorto the substrate is critical because the receptor must have access tothe factor. Furthermore, for some growth factors, dimerization oraggregation in the membrane is believed to be critical, as described in“Peptide Growth Factors and Their Receptors I” M. B. Sporn and A. B.Roberts, eds. (Springer-Verlag. New York. 1990). Thus, the growth factorwill either have to be immobilized in extremely high concentration orimmobilized on flexible tethers which will allow the ligand-receptorcomplex to aggregate in the cell membrane. Direct immobilization of evenhigh concentrations of growth factor may be ineffective if the receptorsbind randomly.

[0016] Tethers

[0017] Requirements

[0018] As used herein, a tether is a flexible link between an attachmentsubstrate and a growth effector molecule. Flexible tethers for attachinggrowth effector molecules to a substrate must satisfy two importantrequirements: (1) the need for mobility of the ligand-receptor complexwithin the cell membrane in order for the effector molecule to exert aneffect, and (2) biocompatibility of materials used for immobilization.Substantial mobility of a tethered growth factor is critical becauseeven though the cell does not need to internalize the complex formedbetween the receptor and the growth factor, it is believed that severalcomplexes must cluster together on the surface of the cell in order forthe growth factor to stimulate cell growth. In order to allow thisclustering to occur, the growth factors are attached to the solidsurface, for example, via lone water-soluble polymer chains, which arereferred to as tethers, allowing movement of the receptor-ligand complexin the cell membrane.

[0019] Examples of water-soluble, biocompatible polymers which can serveas tethers include polymers such as synthetic polymers like polyethyleneoxide (PEO), polyvinyl alcohol, polyhydroxyethyl methacrylate,polyacrylamide, and natural polymers such as hyaluronic acid,chondroitin sulfate, carboxymethylcellulose, and starch.

[0020] Tethers can also be branched to allow attachment of multiplegrowth effector molecules in close proximity. Branched tethers can beused, for example, to increase the density of growth effector moleculeon the substrate. Such tethers are also useful in bringing multiple ordifferent growth effector molecules into close proximity on the cellsurface. This is useful when using a combination of different growtheffector molecules. Preferred forms of branched tethers are star PEO andcomb PEO.

[0021] Star PEO is formed of many PEO “arms” emanating from a commoncore. Star PEO has been synthesized, for example, by living anionicpolymerization using divinylbenzene (DVB) cores, as described by Gnanouet al., Makromol. Chemie 189: 2885-2892 (1988), and Merrill, J.Biomater. Sci. Polymer Edn 5: 1-11 (1993). The resulting molecules have10 to 200 arms, each with a molecular weight of 3,000 to 12,000. Thesemolecules are about 97% PEO and 3% DVB by weight. Other core materialsand methods may be used to synthesize star PEO. Comb PEO is formed ofmany PEO chains attached to and extending from the backbone of anotherpolymer, such as polyvinyl alcohol. Star and comb polymers have theuseful feature of grouping together many chains of PEO in closeproximity to each other.

[0022] Length

[0023] The length of a tether is limited only by the mechanical strengthof the tether used and the desired stability of a tethered growthfactor. It is expected that stronger tethers can be made longer thanweaker tethers, for example. It is also desirable for tether length andstrength to be matched to give a desired half life to the tether, priorto breakage, and thereby adjust the half life of growth factor action.The minimum tether length also depends on the nature of the tether. Amore flexible tether will function well even if the tether length isrelatively short, while a stiffer tether may need to be longer to alloweffective contact between a cell and the growth effector molecules.

[0024] The backbone length of a tether refers to the number of atoms ina continuous covalent chain from the attachment point on the substrateto the attachment point of the growth effector molecule. All of thetethers attached to a given substrate need not have the same backbonelength. In fact, using tethers with different backbone lengths on thesame substrate can make the resulting composition more effective andmore versatile. In the case of branched tethers, there can be multiplebackbone lengths depending on where and how many growth effectormolecules are attached. Preferably, tethers can have any backbone lengthbetween 5 and 50,000 atoms. Within this preferred range, it iscontemplated that backbone length ranges with different lower limits,such as 10, 15, 25, 30, 50, and 100, will have useful characteristics.

[0025] Such tethers are not intended to be limited by the manner inwhich the substrate-tether-growth effector molecule composition isassembled. For example, if linker molecules are attached to thesubstrate and the growth effector molecule, and then the linkers arejoined to form the tethered composition, the entire length of the joinedlinkers is considered the tether. As another example, the attachmentsubstrate may, by its nature, have on its surface protruding molecularchains. If a linker molecule is attached to the substrate via suchprotruding chains, then the chain and linker together are considered tobe a tether.

[0026] Biocompatible polymers and spacer molecules are well known in theart and most are expected to be suitable for forming tethers. The onlyimportant characteristics are biocompatibility and flexibility. That is,the tether should not be made of a substance that is cytotoxic or, inthe case of in vivo uses, which causes significant allergic or otherphysiological reaction when implanted. The tether should also allow thegrowth factor a sufficient range of motion to effectively bind to a cellsurface receptor.

[0027] The biodegradability of a tether, the tether-substrate link, orthe tether-growth factor link can be used to regulate the length of timea growth factor stimulates growth. For example, if a given tetherdegrades during cell growth at a consistent rate, then a limit can beplaced on how long the growth factors binds to and stimulates cellgrowth. Once untethered, a growth factor can be internalized by the cellor can diffuse away from the target cells. Such planned degradation isespecially useful in the context of implanted compositions, used tostimulate tissue replacement, by limiting the amount of tissue growth.

[0028] Attachment Substrates

[0029] There are two basic types of substrates onto which growtheffector molecules can be tethered. One class includes biocompatiblematerials which are not biodegradable, such as polystyrenes,polyethylene vinyl acetates, polypropylenes, polymethacrylates,polyacrylates, polyethylenes, polyethylene oxides, glass, polysilicates,polycarbonates, polytetrafluoroethylene, fluorocarbons, nylon, siliconrubber, and stainless steel alloys. The other class of materialsincludes biocompatible, biodegradable materials such as polyanhydrides,polyglycolic acid, polyhydroxy acids such as polylactic acid,polyglycolic acid, and polylactic acid-glycolic acid copolymers,polyorthoesters, polyhydroxybutyrate, polyphosphazenes,polypropylfumerate, and biodegradable polyurethanes, proteins such ascollagen and polyamino acids, and polysaccharides such asglycosaminoglycans, alginate, and carageenan, bone powder orhydroxyapatite, and combinations thereof. These biodegradable polymersare preferred for in vivo tissue growth scaffolds. Other degradablepolymers are described by Engleberg and Kohn, Biomaterials 12: 292-304(1991).

[0030] Attachment substrates can have any useful form including bottles,dishes, fibers, woven fibers, shaped polymers, particles andmicroparticles. For in vitro cell growth, the growth effector moleculecan be tethered to standard tissue culture polystyrene petri dishes.Woven fibers are useful for stimulating growth of tissue in the form ofa sheet, sponge or membrane.

[0031] The biodegradability of a substrate can be used to regulate thelength of time the growth factor stimulates growth and to allowreplacement of implanted substrate with new tissue. For this purpose thesubstrate with tethered growth effector molecules can be considered ascaffold upon which new tissue can form. As such, a degradable scaffoldis broken down as tissue replacement proceeds. Once released from thesubstrate, a growth factor can be internalized or can diffuse away fromthe target cells. Such planned degradation is especially useful in thecontext of implanted compositions, used to stimulate tissue replacement,by limiting the amount of tissue growth and eliminating the need toremove the tissue scaffold. For implantation in the body, preferreddegradation times are typically less than one year, more typically inthe range of weeks to months.

[0032] In some embodiments, attachment of the cells to the substrate isenhanced by coating the substrate with compounds such as extracellularmembrane components, basement membrane components, agar, agarose,gelatin, gum arabic, collagen types I, II, III, IV, and V, fibronectin,laminin, glycosaminoglycans, mixtures thereof, and other materials knownto those skilled in the art of cell culture.

[0033] Growth Effector Molecules

[0034] Growth effector molecules, as used herein, refer to moleculesthat bind to cell surface receptors and regulate the growth, replicationor differentiation of target cells or tissue. Preferred growth effectormolecules are growth factors and extracellular matrix molecules.Examples of growth factors include epidermal growth factor (EGF),platelet-derived growth factor (PDGF), transforming growth factors(TGFα, TGFβ), hepatocyte growth factor, heparin binding factor,insulin-like growth factor I or II, fibroblast growth factor,erythropoietin, nerve growth factor, bone morphogenic proteins, musclemorphogenic proteins, and other factors known to those of skill in theart. Additional growth factors are described in “Peptide Growth Factorsand Their Receptors I” M. B. Sporn and A. B. Roberts, eds.(Springer-Verlag, New York, 1990), for example.

[0035] Growth factors can be isolated from tissue using methods know tothose of skill in the art. For example, growth factors can be isolatedfrom tissue, produced by recombinant means in bacteria, yeast ormammalian cells. For example. EGF can be isolated from the submaxillaryglands of mice and Genentech produces TGF-β recombinantly. Many growthfactors are also available commercially from vendors, such as SigmaChemical Co, of St. Louis, Mo., Collaborative Research, Genzyme,Boehringer, R&D Systems, and GIBCO, in both natural and recombinantforms.

[0036] Examples of extracellular matrix molecules include fibronectin,laminin, collagens, and proteoglycans. Other extracellular matrixmolecules are described in Kleinman et al. (1987) or are known to thoseskilled in the art. Other growth effector molecules useful for tetheringinclude cytokines, such as the interleukins and GM-colony stimulatingfactor, and hormones, such as insulin. These are also described in theliterature and are commercially available.

[0037] The specific function or effect of a growth effector moleculedoes not limit its usefulness in the disclosed compositions and methods.This is because tethering of a growth effector molecule is used toprevent loss of effect caused by diffusion away from a target celland/or internalization of a growth factor.

[0038] Only those growth effector molecules that can exert an effectwhile tethered are useful in the disclosed compositions. Such an effect,however, need not be the same effect or require the same concentrationas the untethered growth effector molecule. So long as a growth effectormolecule can exert any desired growth effect on a cell while tethered itis considered to be useful for tethering. These useful effects can bedetermined by tethering a selected growth effector molecule andobserving the effect on cell growth using growth assays, such as thosedescribed in the examples below.

[0039] Attachment Methods

[0040] Standard immobilization chemistries, which are well known in theart, can be used to covalently link the tethers to the growth effectormolecule and the substrate. Tethering growth effector molecules can beaccomplished by attachment, for example, to aminated surfaces,carboxylated surfaces or hydroxylated surfaces using standardimmobilization chemistries. Examples of attachment agents are cyanogenbromide, succinimide, aldehydes, tosyl chloride, avidin-biotin,photocrosslinkable agents, epoxides and maleimides. A preferredattachment agent is glutaraldehyde. These and other attachment agents,as well as methods for their use in attachment, are described in“Protein immobilization: fundamentals and applications” Richard F.Taylor, ed. (M. Dekker, New York. 1991). Growth effector molecules canbe tethered to a substrate by chemically cross-linking a tether moleculeto reactive side groups present within the substrate and to a free aminogroup on the growth effector molecule. For example, synthetic EGF may bechemically cross-linked to a substrate that contains free amino orcarboxyl groups using glutaraldehyde or carbodiimides as cross-linkeragents. In this method, aqueous solutions containing free tethersmolecules are incubated with the substrate in the presence ofglutaraldehyde or carbodiimide. For crosslinking with glutaraldehyde thereactants can be incubated with 2% glutaraldehyde by volume in abuffered solution such as 0.1 M sodium cacodylate at pH 7.4. Otherstandard immobilization chemistries are known by those of skill in theart and can be used to join substrates, tethers, and growth effectormolecules.

[0041] For the disclosed cell growth compositions, growth effectormolecules may be tethered either alone or in combinations. For example,both insulin and EGF may be tethered to the same substrate. The growtheffector molecules may be combined in any desired proportions. Therelative amounts of different growth effector molecules can becontrolled, for example, by first separately linking the growth effectormolecules to tethers, then mixing the “loaded” tethers in the desiredproportions and attaching them to the substrate. The proportion of eachgrowth effector molecule tethered to the substrate should match theproportion of loaded tethers in the attachment reaction.

[0042] Tethering to Aminated Surfaces.

[0043] Cell culture surfaces bearing primary amines can be prepared, forexample, by amino-siloxane treatment of glass using reagents which canbe commercially purchased and applied to standard laboratory glasswareor by plasma discharge treatment of polymers in an ammonia environment.Collagen matrices for tissue regeneration have primary amines present inlysine side chains and the terminal amines of each molecule. Twoapproaches are possible. Polymers such as PEO tethers can be activatedon both ends with a leaving group such as tresyl chloride which reactswith primary amines. No blocking is necessary because only the terminalhydroxyl residues of tether are reactive. This type of reaction can becarried out using standard glassware in a chemical fume hood. Vacuumdrying of the product is required as an intermediate step. A substantialexcess of the activated tether over the number of available amines,dissolved in a saline buffer, is added to the surface to be modified andthe coupling reaction is allowed to proceed. Use of an excess ofactivated PEO in this step minimizes the reaction of both ends of PEOwith available amines and ensure a substantial fraction of unreactedactivated chain ends are left for reaction with the growth factor.Unreacted PEO is washed away, and the EGF is then added in salinesolution to react with the remaining activated chain ends. If mouse EGFis used, only the terminal amino acid is reactive because it contains noother primary amines. Human EGF contains three possible immobilizationsites. After the reaction is completed, excess unreacted growth factoris removed. This first approach is preferred for attaching EGF to amatrix such as crosslinked collagen, which contains a large number offree hydroxyls and which does not allow significant non-specificadsorption of EGF.

[0044] A second approach is to activate the tethers on only one endinitially by using a substoichiometric amount of activating agent. Thiswill yield a distribution of species which include completelyunactivated tether as well as tether activated at both ends. Unactivatedtether can easily be washed away after the attachment step. The tetheris then coupled to the support as described above, and the free tetherends are then activated to allow attachment of EGF. This second approachis preferred for derivatization of cell culture surfaces, which mightallow substantial non-specific adsorption of growth factor, because anintermediate step in which unreacted amines are blocked with short-chainmonomethoxy PEO can be added before EGF attachment in order to minimizenon-specific adsorption of the factor.

[0045] Cells

[0046] Cells to be cultured using the disclosed compositions can be anycells that respond to growth factors or that need growth effectormolecule for growth. For example, cells can be obtained from establishedcell lines or separated from isolated tissue. Cells types that can beused with the tethered growth effector molecule compositions includemost epithelial and endothelial cell types, for example, parenchymalcells such as hepatocytes, pancreatic islet cells, fibroblasts,chondrocytes, osteoblasts, exocrine cells, cells of intestinal origin,bile duct cells, parathyroid cells, thyroid cells, cells of theadrenal-hypothalamic-pituitary axis, heart muscle cells, kidneyepithelial cells, kidney tubular cells, kidney basement membrane cells,nerve cells, blood vessel cells, cells forming bone and cartilage, andsmooth and skeletal muscle. The cells used can also be recombinant.Methods for gene transfer are well known to those skilled in the art.

[0047] In Vitro Cell and Tissue Growth Using Substrates with TetheredGrowth Effector Molecules

[0048] Substrates with tethered growth effector molecules can be used toimprove in vitro culture of hard-to-grow cells such as liver cells.Liver cell cultures would be useful for toxicology testing to replacecertain aspects of animal testing of drugs. Liver cells grow very poorlyin vitro using prior art methods, typically undergoing only one or tworounds of DNA synthesis after they are placed in culture. Since atethered growth factor cannot be internalized, tethering will change theway the cells respond to the factor, constantly stimulating them togrow.

[0049] Cells can be cultured with tethered growth effector moleculecompositions using any of the numerous well known cell culturetechniques. Standard cell culture techniques are described in Freshney,“Cell Culture, a manual of basic technique” Third Edition (Wiley-Liss,New York, 1994). Other cell culture media and techniques well known tothose skilled in the art can be used with the disclosed compositions.The disclosed compositions are adaptable to known cell culture vessels.For example, growth effector molecules can be immobilized on standardtissue culture polystyrene and glass petri dishes, T-flasks, rollerbottles, stackable chambers, and filter systems such as the MilliporeMILLICELL™ inserts, hollow fiber reactors and microcarriers. Cells canalso be cultured in suspension using the disclosed compositons bytethering growth effector molecules to tiny beads or fibers, on theorder of 10 microns in diameter of length. Such tiny particles, whenadded to culture medium, would attach to cells thereby stimulating theirgrowth and providing attachment signals. The only critical difference inculturing technique is the elimination of growth factor from the cellculture medium when using tethered growth factor compositions. Asdescribed in the examples below, using soluble versus tethered EGF inprimary hepatocyte cultures show an enhanced DNA synthesis rate of thetethered growth factor in comparison to the soluble growth factor. Thiseffect is dependent on the amount of the immobilized factor.

[0050] In Vivo Tissue Growth Using Tissue Growth Scaffolds with TetheredGrowth Effector Molecules

[0051] In yet another embodiment of the present invention, erodible andnon-erodible artificial matrices with tethered growth effector moleculesmay be used either alone or in combination with attached cells toremodel tissue architecture or to repair tissue defects and wounds.

[0052] Known methods and compositions for culturing cells and implantingthem into the body can be adapted to use tethered growth effectormolecules. For example, U.S. Pat. No. 4,352,883 to Lim, uses cells thatare encapsulated within alginate microspheres, then implanted. Suchmicrospheres can be modified with tethered growth effector molecules toimprove their usefulness. Culturing cells on a matrix for use asartificial skin, as described by Yannas and Bell in a series ofpublications, can also be modified by tethering growth effectormolecules to the matrix. U.S. Pat. No. 4,485,097 to Bell, U.S. Pat. No.4,060,081 to Yannas et al., and U.S. Pat. No. 4,458,678 to Yannas et al.describe substrates for use as artificial skin. U.S. Pat. No. 4,520,821to Schmidt describes a similar approach that was used to make linings torepair defects in the urinary tract.

[0053] Vacanti et al., Arch. Surg. 123: 545-549 (1988), describes amethod of culturing dissociated cells on biocompatible, biodegradablematrices for subsequent implantation into the body. Cima and Langer,“Tissue Engineering” Chem. Eng. Prog. 89: 46-54 (1993), describeimportant considerations for the nature and form of implanted matricesuseful for inducing tissue replacement. U.S. patent application Ser. No.08/200,636 entitled “Tissue Regeneration Matrices by Solid Free ForceFabrication” filed Feb. 23, 1994 by Cima and Cima, which is herebyincorporated by reference, describes tissue regeneration matrices,fabrication techniques, and methods of regenerating tissue. In general,tissue regeneration devices can be constructed from polymers, ceramics,or from composites of ceramics and polymers. Common materials useful forconstructing tissue regeneration devices are, for example, extracellularmatrix proteins, especially collagens; degradable polyesters, such aspolylactic acid, polyglycolic acid, co-polymers of polylactic acid andpolyglycolic acid, and polycapralactone; polyhydroxybutyrate;polyanhydrides; polyphosphazenes; bone powder; natural polysaccharides,such as hyaluronic acid, starch, and alginate; hydroxyapatite;polyurethanes; and other degradable polymers described by Engleberg andKohn, Biomaterials 12: 292-304 (1991). All of these known compositionscan be modified by tethering growth effector molecules to the substrate.

[0054] Growth effector molecule tethered compositions for in vivo usecan be in the form of polymeric, attachment molecule-coated sutures,pins, wound dressings, fabric, and space-filling materials. Attachmentsubstrates that promote ingrowth of dermal fibroblasts and capillariescould also be used for dermatological applications and cosmetic surgery,such as repair of wrinkles and aging skin, burn therapy, or skinreconstruction following disfiguring surgery. Substrates with tetheredgrowth effector molecules that promote osteoblast migration could beused to fill bone defects following tumor surgery or for non-healingfractures. Substrates with tethered growth effector molecules thatpromote muscle cell growth and migration could be used for replacementof muscle mass, including cardiac muscle and smooth muscle, followingdisfiguring surgery and for patients with muscle degeneration ordysfunction. Tubular substrates with tethered growth effector moleculesthat promote growth, migration, and function of epithelial, endothelialand mesenchymal cells can be used for construction of artificial ductsfor carrying bile, urine, gases, food, semen, cerebrospinal fluid,lymph, or blood. Sheaths formed of substrates that promote growth offibroblasts from perichondrium, periosteum, dura mater, and nervesheaths may be used to recreate these structures when they are injuredor lost due to surgery or cancer. In all of these embodiments, eitherthe substrate with tethered growth effector molecules or substrate plusattached cells may be used for reconstruction in vivo.

[0055] Substrates for promoting tissue generation can be formed to havea desired tissue shape. As used herein, a desired tissue shape is theshape that the newly generated tissue is desired to have. For example,certain tissues may need to be sheet-like, tubular, or formed as a lobe.

[0056] Deactivation of the growth factor once appropriate tissueregeneration has occurred can be accomplished by tethering the growthfactor to a support which slowly degrades. Examples of such supportmaterials are polylactide-co-glycolide and crosslinked hyaluronic acidor collagen. Properly shaped substrate with tethered growth effectormolecules can be applied in clinical problems such as healing of skin orperiodontal ligament by encouraging continued tissue growth for the lifeof a shaped, degradable implant.

[0057] The disclosed compositions can be administered to animals invarious modes, including implantation, injection, and infusion. Knownimplantation techniques can be used for delivery of many different celltypes to achieve different tissue structures. The tethered growtheffector molecule compositions may be implanted in many different areasof the body to suit a particular application.

[0058] Drug and Toxicity Testing Using Tissue Grown In Vitro on TetheredSubstrates

[0059] In another embodiment, cells are cultured on substrates withtethered growth effector molecules and the resulting cell cultures areused to screen compounds for effects on cell growth, cell proliferation,cell metabolism, and DNA. For example, the cultured cells can be used toscreen for compounds that alter hepatocyte enzyme systems. The culturedcells can also be used to study metabolism of various compounds and thecarcinogenicity or mutagenicity of compounds both before and aftermetabolism by the cells.

[0060] Classically, compounds have been assayed for mutagenic activityusing short term tests (STT) employing bacterial cell systems or animalstudies. Most animal studies are conducted using the protocol forrodents developed by the National Cancer Institute in the early 1970sand reported by Sontag et al. in U.S. Dep. Health Educ. Welfare Publ.(NIH) Carcinog. Tech. Rep. Serv. 1: 76 (1976). Four STTs that areroutinely used are the Salmonella mutagenesis, SAL, described by Haworthet al., Environ. Mutagen. 5 (suppl. 1): 3 (1983) and Mortelmans et al.Toxicol. Appl. Pharmacol. 75: 137 (1984); chromosome aberrations inChinese hamster ovary cells, ABS; sister chromatid exchanges in Chinesehamster ovary cells, SCE, both described by Galloway et al. Environ.Mutagen. 7: 1 (1985); and mouse lymphoma cell, MOLY, assays, describedby Myhr et al., “Evaluation of Short-Term Tests for Carcinogens: Reportof the International Programme on Chemical Safety's Collaborative Studyon in vitro Assays” vol. 5 of Progress in Mutation Research Series,pages 55-568, Ashby et al., Editors (Elsevier, Amsterdam, 1985).Unfortunately, the correlation between the rodent assays and the STTs ispoor, and the available STTs do not provide a method for testingcompounds for toxicity or mutagenicity of normal organ-specific cells,nor the effect of metabolism on the compounds by the organ-specificcells, such as hepatocytes.

[0061] When testing the effect of potential toxins, control assays usingknown toxins are used for comparison. Examples of known hepatotoxins,such as acetaminophen, carbon tetrachloride, alcohol, and cell-specificviruses such as hepatitis viruses, can be used to test the suitabilityof the model tissue. Standard cell number or cell lysis assays, such asLactate dehydrogenase release, can be used to measure toxicity. Numerousother toxicity and mutagenesis assays are known in the art and can bepracticed using cell cultures grown on the tethered growth effectormolecule substrates described herein.

[0062] The disclosed compositions can be used to grow liver cells invitro and make it feasible to use in vitro liver cell cultures to carryout biotransformations by applying the compound of interest directly toliver cells in culture. The supernate from the liver cell cultures canthen be applied to other types of cells, such as skin, lung, nerve, andbladder, to assess any derived effect of the compound of interest. Anautomated system which pumps culture medium through a liver cell cultureand then to cultures of these other cell types can be used.

[0063] The present invention is further understood by reference to thefollowing non-limiting.

EXAMPLE 1 Enhancement of Cell Growth

[0064] Cell Growth and Cell Growth Assessment Methods

[0065] A. In Vitro Hepatocyte Culture System.

[0066] Rat hepatocytes were prepared according to Cima et al.,Biotechnology and Bioengineering 38: 145-158 (1991). Briefly, rat liverswere perfused with calcium-free perfusion buffer followed by perfusionbuffer with CaCl₂ and collagenase until the livers became soft. Cellswere dispersed in William's Medium E supplemented with 10 ng/mL EGF(Collaborative Research), 20 mM pyruvate (Gibco), 5 nM dexamethasone(Sigma), 20 mU/mL insulin (Gibco), 100 U/mL Penicillin/Streptomycin(Gibco). Cells were grown in culture generally as described by Cima etal. (1991). Briefly, cells were plated in culture medium at aconcentration of 3×10⁴ viable cells per square centimeter of culturesurface area. Following attachment, the medium was changed to removeunattached cells and then cells were maintained in medium with dailymedium changes. The base culture medium for growth on tetheredsubstrates and control substrates was William's Medium E supplementedwith 0.55 g/L sodium pyruvate. 0.5 pM dexamethasone. 0.8 mg/mL insulin(bovine). 100 U/mL Penicillin/Streptomycin, and 2 mM L-glutamine. Insome cases, the medium was supplemented with EGF.

[0067] B. Quantitative Dot-Blot Assay.

[0068] Secretion rates for the proteins albumin, transferrin,fibrinogen, and fibronectin from the hepatocyte cultures were measuredwith a quantitative dot-blot assay. Media samples from the cultures wereserially diluted and loaded in duplicate onto nitrocellulose paper with0.1 micron pore size using a 96 well minifold apparatus(Schleicher-Schuell). Protein standards were also loaded in duplicate atdecreasing levels from 300 to 10 ng/dot. The blot was then exposed to anappropriate primary antibody for the protein being quantitated. Rabbitanti-rat albumin and anti-rat transferrin were available from Cappel.Rabbit anti-rat fibrinogen was available from Sigma. The non-boundprimary antibody was washed away after one hour, and the blot wasexposed to donkey anti-rabbit IgG labelled with ¹²⁵I (Amersham) for anadditional hour. The non-bound secondary antibody was washed away, andan autoradiograph of the blot was made. The dots were then punched outand bound ¹²⁵I measured using a gamma counter to determine the totalamount of bound antibody. A calibration curve was generated by relatingknown amounts of standard protein to total count per minute bound. Thelinear portion of the standard curve was then used to quantitate theamount of protein in the unknown media samples. Secretion rates werenormalized for cell number before the modulating effects of differentattachment molecule densities were compared.

[0069] C. One Dimensional SDS-PAGE of Secreted Proteins.

[0070] The pattern of protein secretion from cultured hepatocytes wasdetermined by pulse labelling cultures from 46 to 48 hourspost-attachment with ³⁵S labelled methionine (ICN) in methionine freeWilliam's E media (Gibco), with or without EGF. The media was collectedafter the two hour labelling, and equal amounts of protein were analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Autoradiographs were prepared with XOMAT-XAR5 film.

[0071] D. DNA Synthesis Measurement.

[0072] DNA synthesis is used as a measure of potential for cellularproliferation. Hepatocytes were pulse labelled for 20 hours beginning at48 hours post-cell attachment with bromedeoxyuridine (BrdU), andsubsequently fixed as outlined above. Cells were processed forimmunocytochemistry using a BrdU kit from Amersham. Briefly, nuclei werepermeabilized with DNAse I during incubation with the primary antibody.Detection of the bound antibody was achieved using peroxidase conjugatedantibody to mouse immunoglobulin, and polymerizing diaminodenzidine(DAB) in the presence of cobalt and nickel, giving black staining atsites of BrdU incorporation. Alternatively, hepatocytes are pulselabelled for 16 hours beginning at 48 hours post-cell attachment with³H-thymidine, and subsequently fixed in 95% ethanol/5% acetic acidfixative for several hours. The dishes or slides were coated with KodakNTB2 autoradiography emulsion, and allowed to expose for seven days.Autoradiographic grains were developed using Kodak D-19 developer. Thepercentage of cells actively synthesizing DNA was quantitated bychoosing 8 random areas on each dish and counting those cells withlabelled nuclei versus the total number of cells. A minimum of 35 cellswas counted per dish.

[0073] Synthesis of Growth Substrate using Polyethylene Oxide Tether

[0074] A. Silyation Reaction.

[0075] Glass microscope slides were cleaned by immersion in 1:1methanol:HCl for at least 30 minutes. They were rinsed twice in waterand immersed in 1:1 water:concentrated sulfuric acid for at least 30minutes. After another rise in water, the slides were placed in boilingwater for 15 to 30 minutes. In a glove box under a nitrogen atmosphere,the freshly cleaned slides were placed in a solution of freshly mixedacidic methanol (1.0 mM acetic acid in methanol), 5.0% H₂, and 1% ETDA(N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane) for 15 minutes, andthen rinsed three times in methanol. Following the final rinse theslides were baked on a 120° C. oven for 5 to 10 minutes. The slides werestored in a desiccator at room temperature while awaiting polymergrafting.

[0076] B. Activation of Polymer.

[0077] Star polyethylene oxide was dissolved in methylene chloride (10wt %) and dried over molecular sieve at 4° C. 110 microliters drytriethylamine and 75 microliters tresyl chloride were added to the drypolymer solution for every gram of polymer. After 90 minutes the solventwas evaporated under vacuum and the polymer was redissolved in acidifiedmethanol (0.06 M HCl in methanol) and allowed to precipitate at −20° C.To remove unreacted tresyl chloride, the polymer was reprecipitated sixtimes, after which the solvent was evaporated and the dried activatedpolymer stored under nitrogen.

[0078] C. PEO Grafting and Re-Activation.

[0079] Slides were grafted with star polyethylene oxide by placing adroplet of 0.1 to 10 wt % tresyl chloride activated polymer in 0.1 Mphosphate buffer (pH 7.4) on each slide and allowing the reaction toproceed for 12 hours. The slides were rinsed in phosphate buffer andthen in water. Slides were dried in graded ethanol solutions of,sequentially, 25%, 50%. 75% S, and 100% ethanol. Then the slides wererinsed in dry acetone and finally in dry methylene chloride beforereactivation. To tresyl activate the grafted star PEO, slides wereimmersed for 1 hour in 0.06 M tresyl chloride. 0.07 M triethylamine inmethylene chloride at room temperature under a dry nitrogen atmosphere.For mock activation controls, the tresyl chloride was omitted.

[0080] D. EGF Coupling and Desorption.

[0081]¹²⁵-EGF of murine origin was coupled to activated slides in 0.01 Mphosphate buffer (pH 7.4) for 12 hours at room temperature. The sameprocedure was followed for control slides. Adsorbed EGF was desorbed bysuccessive washes in 0.01 M phosphate buffer (pH 7.4) with 0.1 wt %bovine collagen. The amount of EGF associated with the slides wasdetermined using a gamma counter. The amount of EGF coupled to activatedslides was determined by subtracting the amount adsorbed to the controlslides from that associated with the activated slides.

[0082] Growth of Cells on Tethered Substrate In Vitro

[0083] Freshly isolated rat hepatocytes were seeded on tethered EGFslides and control slides, prepared as described above. The seededslides were incubated in William's Medium E supplemented with 0.55 g/Lsodium pyruvate, 0.5 pM dexamethasone, 0.8 mg/mL insulin (bovine), 100U/mL Penicillin/Streptomycin, and 2 mM L-glutamine. Cells were labelledwith bromedeoxyuridine (BrdU) as described above.

[0084] Cell seeding and DNA synthesis assays were performed on slidesthat had been activated with tresyl chloride and coupled with EGF, onmock activated control slides, both prepared as described above, and oncontrol tissue-culture treated polystyrene dishes (Falcon) either withEGF added to the medium at 10 ng/mL or with EGF omitted.

[0085] The results of the DNA synthesis assay for the latter,non-tethered controls is shown in FIG. 1. The presence of EGF in themedium clearly causes an increase in the number of cells synthesizingDNA. The results of the DNA synthesis assay for the tethered EGF surfaceand the mock activated control surface that had only adsorbed EGF isshown in FIG. 2. The number of cells synthesizing DNA is clearly higherfor the tethered EGF surface.

[0086] Modifications and variations of the compositions and methods ofthe present invention will be obvious to those skilled in the art fromthe foregoing detailed description. Such modifications and variationsare intended to come within the scope of the appended claims.

1-13. (canceled)
 14. The method of claim 33 wherein the attachment agentis selected from the group consisting of cyanogen bromide, succinimide,aldehyde, tosyl chloride, avidin-biotin, epoxide, maleimide, andcarbodiimide.
 15. The method of claim 14 wherein the composition isadministered by injection, infusion, or implantation.
 16. The method ofclaim 15 wherein the composition is administered by implantation of thecomposition and wherein the substrate is shaped to match a desiredtissue shape.
 17. The method of claim 16 wherein the substrate isbiodegradable. 18-31. (canceled)
 32. The method of claim 34 wherein theattachment agent is selected from, the group consisting of cyanogenbromide, succinimide, aldehyde, tosyl chloride, avidin-biotin, epoxide,maleimide, and carbodiimide.
 33. A method for growing eukaryotic cellscomprising bringing into contact the cells with a composition comprisinga biocompatible solid substrate, biocompatible polymeric tethers, andgrowth effector molecules, wherein one end of each tether is covalentlylinked to the substrate and one end is covalently linked to a growtheffector molecule so that the growth effector molecule cannot beinternalized by cells attached to the substrate; wherein the growtheffector molecules are attached to the substrate in a concentrationeffective to enhance the rate of target cell growth over the rate oftarget cell growth with soluble growth effector molecules and growtheffector molecules adsorbed to a substrate, without internalization ofthe molecules; and wherein the tether is covalently linked to thesubstrate and to the growth effector molecule by the same attachmentagents, maintaining the cells in contact with the composition underconditions and for a time sufficient to cause the cells to grow.
 34. Amethod of testing a compound for an effect on tissue comprising bringinginto contact the compound to be tested and a composition comprising abiocompatible solid substrate, biocompatible, polymeric tethers, growtheffector molecules, and growing cells, wherein one end of each tether iscovalently linked to the substrate and one end is covalently linked to agrowth effector molecule so that the growth effector molecule cannot beinternalized by cells attached to the substrate; wherein the growtheffector molecules are attached to the substrate in a concentrationeffective to enhance the rate of target cell growth over the rate oftarget cell growth with soluble growth effector molecules and growtheffector molecules adsorbed to a substrate, without internalization ofthe molecules; where the tether is covalently linked to the substrateand to the growth effector molecule by the same attachment agents; andwherein the growing cells are bound to the growth effector molecules;incubating the compound and the composition under promoting cell growth;and observing the cells for any effect not observed in cells not broughtinto contact with the composition.